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Molecular characterization and antibacterial effect of endophytic
actinomycetes Nocardiopsis sp. GRG1 (KT235640) from brown algae
against MDR strains of uropathogens
Govindan Rajivgandhi
a
, Ramachandran Vijayan
a
,
b
, Marikani Kannan
c
,
Malairaja Santhanakrishnan
d
, Natesan Manoharan
a
,
*
a
Department of Marine Science, Bharathidasan University, Tiruchirappalli 24, Tamil Nadu, India
b
School of Life Sciences, Jawaharlal Nehru University, New Delhi 67, India
c
Department of Microbiology, VHNSN College, Virudunagar 01, Tamil Nadu, India
d
Department of Marine and Coastal Studies, Madurai Kamaraj University, Madurai 21, Tamil Nadu, India
article info
Article history:
Received 31 August 2016
Accepted 10 November 2016
Available online xxx
Keywords:
Endophytes
Multi-drug resistant strains
Urinary tract infections
Minimal inhibitory concentration
abstract
Our study is to evaluate the potential bioactive compound of Nocardiopsis sp. GRG1 (KT235640) and its
antibacterial activity against multi drug resistant strains (MDRS) on urinary tract infections (UTIs). Two
brown algae samples were collected and were subjected to isolation of endophytic actinomycetes. 100
strains of actinomycetes were isolated from algal samples based on observation of morphology and
physiological characters. 40 strains were active in antagonistic activity against various clinical pathogens.
Among the strains, 10 showed better antimicrobial activity against MDRS on UTIs. The secondary
metabolite of Nocardiopsis sp. GRG1 (KT235640) has showed tremendous antibacterial activity against
UTI pathogens compared to other strains. Influence of various growth parameters were used for syn-
thesis of secondary metabolites, such as optimum pH 7, incubation time 5e7 days, temperature (30
C),
salinity (5%), fructose and mannitol as the suitable carbon and nitrogen sources. At 100
m
g/ml concen-
tration MIC of Nocardiopsis sp. GRG1 (KT235640) showed highest percentage of inhibition against Proteus
mirabilis (85%), and E.coli, Staphylococcus auerues, Psuedomonas aeroginasa, Enterobactor sp and Coaguli-
nase negative staphylococci 78e85% respectively.
©2016 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
1. Introduction
Urinary tract infections (UTIs) are the bacterial infection that
affects of all age group to any part of the urinary tract. It is the
second most common infectious presentation in community
medical practice. It affects the group of all age people. 50 Million
People are affected by UTI each year in all over the world [1]. The
risk of developing UTI infection in diabetic patients is higher. Dia-
betic cystopathy and micro-vascular diseases may cause changes in
host defense mechanism of kidney leads to higher incidence of UTI
[2]. UTIs patients are susceptible to cause emphysematous cystitis,
pyelonephritis, renal or perinephric abscess, bacteremia, and renal
papillary necrosis. Bacteraemic patients have more chance to
develop acute renal failure [3].The gram-negative bacteria play an
important role in UTI and the most common causative agent is
Escherichia coli (75e90%) [4,5]. The other gram negative bacterial
pathogens causing UTI are klebsiella sp., Proteus mirabilis and
Pseudomonas aeruginosa. However, the Enterococci and coagulase
negative Staphylococci are the most predominant gram positive
bacteria also present in UTI [6]. Multi drug resistant pathogens are
the major issue in health care industry. In general, the natures of
antibiotic susceptibility of UTI causing pathogens have been
differing from various environmental conditions in both commu-
nity and hospitals surroundings [7,8]. Rapidly growing drug re-
sistances in pathogens are one of the major problems to treat
diseases like malaria, tuberculosis, diarrheal diseases and UTI etc
[9,10].“Some new approaches [11,12] and challenges [13,14] related
to drug resistant pathogens have been recently reported but further
studies are much needed.”In order to identify the novel potential
*Corresponding author. Department of Marine Science, Bharathidasan Univer-
sity, Trichirappalli 24, Tamil Nadu, India.
E-mail address: biomano21@yahoo.com (N. Manoharan).
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Bioactive Materials
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http://dx.doi.org/10.1016/j.bioactmat.2016.11.002
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license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Bioactive Materials xxx (2016) 1e11
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
inhibitor molecules against Multi drug resistant microbes are
essential to eradicate the UTI causing pathogens.
The 70% of the earth surface are made of marine environment.
At this condition, where the microorganisms are synthesized many
different bioactive secondary metabolites. The ocean has an unex-
ploited source for many potential drugs and secondary metabolites
[15]. As marine environment has vast difference than terrestrial
nature. Marine actinomycetes have the capability to synthesize
various types of secondary metabolites at extreme salinity, stress
and temperature. Only marine actinomycetes can able to synthe-
size active compounds against various diseases than terrestrial
nature [16]. Actinomycetes are gram positive, filamentous have the
ability to produce novel bioactive compounds such as antibiotics,
vitamins, herbicides, pesticides, anti-parasitic and enzymes plays a
major role in therapeutic applications and are active against many
pathogenic microorganisms [17,18].
Today, the developing of effective antimicrobial agents is the
major challenge to the health care industry, especially immuno-
compromised patients and multi drug resistant pathogens. Among
the all known actinomycetes especially Streptomyces and Nocar-
diopsis species have been excellent bioactive secondary metabolites
producer of antimicrobial, anti parasitic, antitumor and antiviral
agents [19,20]. The isolation of new compounds by rotation from
terrestrial sources is decreasing because of known compounds
identification. Therefore, it is the urgent need to discover new
group of bioactive metabolites from various marine sources. In
particular, the endophytic actinomycetes from various marine
plants and algae association and its secondary metabolites were not
studied. Hence, our current study was focused on isolation and
characterization of endophytic actinomycetes from brown algae for
screening of antibacterial compounds against multidrug resistant
uropathogens.
2. Materials methods
2.1. Collection of samples
The two young healthy brown macro algae Turbinaria ornata,
and Sargassum wightii were collected from Gulf of Mannar region
(Latitude 9
15'41.88
00
N, Longitude 79
04'05.81
00
E), Rameswaram,
Southeast coast of Tamil Nadu, India. The collected algae samples
were covered by sterile plastic bags to avoid contamination. The
samples were kept in ice box and taken to the laboratory imme-
diately. The collected samples were washed thoroughly with
distilled water for removal of the free floating organisms and epi-
phytes and 70% ethanol was also used for surface sterilization. The
samples were air dried and stored for further study.
2.2. Isolation of endophytic actinomycetes
Isolation and screening of endophytic actinomycetes were
determined by selective media method. The algal samples were
aseptically cut into small pieces (10 mm), and macerated with
sterile distilled water by using mortar and pestle. The macerated
samples were serially diluted up to 10
7
. About 0.1 ml of the
samples was spread on the sterile starch casein agar and actino-
mycetes Isolation Agar (AIA) (HiMedia laboratories Pvt. Mumbai,
India). The plates were incubated at 28 ±2
C for 7e10 days. After
incubation, the growth of endophytic actinomycetes were observed
in the plates and stored in Starch Casein Agar (SCA) medium for
further use [21].
2.3. Validation of endophytic actinomycetes
To prove the isolated actinomycetes were arisen from internal
tissue of the host samples finger prints of the surface sterilized
tissues were validated by using International Strptomyces Project
Medium (ISP) agar plates and incubated for 28
C. The tissues of
algae samples were soaked in water for 2 min with continuous
stirring after the three time distilled water sterilization. The last
wash sample 0.1 ml was taken and inoculated in ISP 2 media. After
incubation, if no microbial growth was observed on the agar plates,
the sterilization was considered as very effective [22].
2.4. Test organisms
The multi drug resistant strains of uropathogens [23,24] such as
E.coli, Proteus mirabilis, Pseudomonas aeroginosa, Klebsiella pneu-
monia, Enterobacter sp., Staphylocoous aereus, Coagulase-negative
Staphylococci, Candida albicanes were obtained from Medical
Microbiology unit, Department of Microbiology, Periyar University,
Salem - 11. The resistances against fourth generation cepholos-
phorin strains were determined by disc diffusion method. The
Collected uropathogens were maintained in glycerol stock and
stored at 20
C for future use.
2.5. Primary screening and antagonistic activity
The Primary screening of antimicrobial activity was determined
by conventional cross streak method [24].The isolated strains were
streaked across the diameter on Muller Hinton Agar (MHA) plates.
The plates were incubated at 28
C for 3e4 days. After observing
the fine growth of the strain, the 24 h cultures of uropathogens
were streaked perpendicular to the angle of central strip of the
actinomycetes culture. All plates were incubated at 37
C for 24 h.
After 24 h, the antagonistic activities of the highly potential strains
were observed based on the zone of inhibition. The broad spectrum
activity of highly potential Nocardiopsis sp. GRG1 (KT235640) cul-
ture filtrate was added with equal volume of five different solvents
(alcohol, dichloromethane, ethyl acetate, chloroform, and meth-
anol) and shaken for 1hr.The antimicrobial activity of extracted
filtrates was performed against test pathogens using well-diffusion
method [25].
2.6. Extraction of bioactive compounds from Nocardiopsis sp. GRG1
(KT235640)
The antimicrobial compounds of Nocardiopsis sp. GRG1
(KT235640) was recovered from the filtrate by active solvent of
ethyl acetate extraction method followed by Ref. [26]. The Nocar-
diopsis sp. GRG1 (KT235640) was inoculated with starch casein
nitrate (SCN-B) broth (50% seawater and 50% distilled water), and
the broth was incubated at 28
C for 7e15 days. After fermentation,
the broth was centrifuged at 10,000 rpm for 10 min and the su-
pernatant was collected and filtered by Whattman No.1 filter paper.
The pellet and cell free supernatant were collected separately for
further use. Ethyl acetate was added with filtrate of supernatant in
the ratio of 1:1(w/v) and shaken vigorously 1hr for complete liquid-
liquid extraction. The organic phase was separated from aqueous
phase were collected using separating funnel and evaporated with
water bath at 40e50
C. After evaporation, the dried crude com-
pounds were collected and determined the antimicrobial activity
against MDRS of UTI pathogens by agar well diffusion method.
2.7. Secondary screening
The antimicrobial activity of Nocardiopsis sp. GRG1 (KT235640)
were performed by against test pathogens [24] at regular intervals
(24 h, 48 h, 96 h) using well-diffusion method [25]. The isolated
[23] UTIs pathogens were spread on MHA plates using sterilized
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e112
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
cotton swab. Each of the plates were cut the well by using gel borer
and five different concentrations viz. 15, 25, 50, 75,100
m
l of the
extract was added separately into each well. The ethyl acetate
extract act as a control. The plates were incubated at 37
C for 24 h.
After 24 h all the plates were observed for the zone of inhibition
around the wells.
2.8. Minimum inhibitory concentration (MIC)
The minimum inhibitory concentrations (MIC) of antimicrobial
compound of Nocardiopsis sp. GRG1 (KT235640) was determined
against MDRs of UTIs pathogen (24) by micro broth dilution
method [27] and the results of MIC was determined by spectro-
photometer using microtiter plate. The various concentration (10,
20, 30, 40, 50, 60, 70, 80, 90, 100
m
g/ml) of ethyl acetate extract of
Nocardiopsis sp. GRG1 (KT235640) was used to identified the MIC
value against MDRS of UTI pathogens. The solution was transferred
into the first well of a 96-well plate (Himedia laboratory, India)
before filled with 100
m
l of tryptone soya broth. The two fold serial
dilution was done in to 11 following wells. 95
m
l of sterilized fresh
tryptone soya broth and 5
m
l of 24 h bacterial culture were added.
The final volume of each well contained100
m
l. The without extract
in the test pathogen of control wells were also prepared. Each plate
was mixed well and then the plates were incubated at 37
C for
24 h. After 24 h, no visible growth was observed in the plate. Lowest
concentrations of the extract were indicated as MIC [28] and the
plates were read with UV spectrophotometer at 570 nm and per-
centage of inhibition was calculated by using the following formula
Percentage of inhibition:
ðControl OD 570 nm Test OD 570 nmÞ
Control OD 570 nm 100
2.9. Biochemical characterization of endophytic actinomycetes
isolates
The biochemical characterization of Indole, MR, VP, Citrate, H
2
S
Urease, Oxidase test with Nocardiopsis sp. GRG1 (KT235640) was
performed by various broth and slant cultures (ST 1). The procedure
was followed by Ref. [34].
2.10. Genomic studies of potential strain
The isolation of Genomic DNA from endophytic actinomycetes
was followed by Ref. [29]. Amplification of 16S rDNA by using
Universal primers. (Actino specific forward Primer -5'-GCCTAACA-
CATGCAAGTCGA-3' and Actino Specific reverse primer - 5'-CGTAT-
TACCGCGGCTGCTGG-5') followed by Ref. [30]. 35 cycles was
performed. Detection of PCR amplification using agarose gel elec-
trophoresis after ethidium bromide staining. Then PCR product was
sent to sequencing by automated sequenced method (Eurofins
Genomics India Pvt Ltd). The same primers as reported above were
used for sequencing. Further, NCBI-BLAST [www.ncbi.nlm.nih.gov/
blast] was used to compare the sequence similarity of isolated
endophytic actinomycete strain with reference actinomycetes
strains. The 16S rDNA a sequence of actinomycetes was deposited in
NCBI and the sequences accession number was obtained. Reference
sequence was downloaded from the Genbank Database (http://
www.ncbi.nlm.nih.gov/genbank). Both the sequences were
aligned by using the multiple sequence alignment program CLUS-
TAL W [31]. The gaps were identified manually from the aligned
sequences and arranged in a block of 250bp in each row and as an
input format in software MEGA V 2.1. The pair wise evolutionary
distances were performed using the Kimura 2-parameter model
[32]. In order to obtain the confidence values for bootstrap analysis,
the original data set was re-sampled toll, 1000 times by using the
bootstrap program of Phylogeny. The bootstrapped data set was
used to build the phylogenetic tree by using the MEGA software.
The resulted multiple distance matrixes was then used to construct
phylogenetic tree using Neighbour Joining method [33].
2.11. Phenotypic characterization
The morphology and biochemical observation of isolated col-
onies are important for taxonomy of actinomycetes. Gram staining,
biochemical characterization, aerial mass color, reverse side
pigment, melanoid pigments, spore chain morphology, and some
minerals such as carbon, nitrogen sources [35,36] were performed
to determine the taxonomy of actinomycetes.
2.11.1. Aerial mass color
The aerial mycelium is one of the important characters for
identification of isolated endophytic actinomycetes. The isolated
strains were inoculated on the starch casein nitrate agar (SCN)
plates and the plates were incubated at 28
C for 6e7 days. After
incubation the nature of the actimycetes studies were observed.
Basically, color of the matured spore forming aerial mycelium is
white, red, grey, blue and violet. Sometimes the aerial mycelium is
also present in combination of two colors. So, both the colors were
also recorded. Sometimes aerial mass color of a strain showed in-
termediate tints, and then also, both the color series should be
noted.
2.11.2. Reverse side pigments
The strains were classified into the following two categories
based on their ability to produce characteristic of pigments on the
reverse side of the colony known as distinctive (Positive) and not
distinctive or none (Negative). Pale yellow of chroma and yellowish
brown color of the growth were recorded as positive (P) and no
color of the plates were recorded as negative (N).
2.11.3. Melanoid pigments
The isolated colonies were inoculated on the ISP-5 plates and
the plates were incubated at 28
C for 4e5 days for identification of
the melonoid pigmentation. After the incubation period, the posi-
tive strains of the cultures showed greenish brown, brown to black
diffusible pigment or a distinct brown pigment modified by other
color are recorded as positive (P).The absence of the pigmentplates
were recorded as negative (N).
2.12. Stress tolerance of endophytic actinomycetes isolates
The identification of stress tolerance observation is most
important for the studies of native strains of actinomycetes. The
ability to check the various stress tolerance (Different concentra-
tion of NaCl, pH, and temperature) of isolated strains (ST 2)were
studied by Ref. [37].
2.12.1. Effect of salinity
Various concentrations of (0, 5, 10,15, 20, 25, 30, 35, 40, 45, 50%)
NaCl solutions were added to the starch casein broth. The actino-
mycetes strains were inoculated into the broth and incubated at
28
C for 7e15 days. After incubation, the positive and negative
growth of the broth was observed and the antimicrobial activity of
the positive growth of the extract was tested against uropathoges.
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e11 3
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
2.12.2. Effect of temperature
The actinomycetes strains were streaked over the actinomycetes
isolating agar plates and the plates were incubated at various
temperatures at 25
C, 30
C, 35
C, 40
C, 45
C, 50
C, 55
C for
7e15 days. After incubation the positive and negative growth of the
plates were identified. Based on the temperature, the potential
activities of the extracts were performed against uropathogens by
agar well diffusion method.
2.12.3. Effect of pH
The pH of the SC broth was adjusted to (4, 5, 6, 7, 8 and 9) with
0.1N NaOH/0.1 N HCl. The entire flask were inoculated with various
strains of endophytic actinomycetes culture and incubated at 28
C
for 7 days. After incubation the positive and negative indication of
the culture were determined. The active strains of the culture were
performed the antimicrobial activity against uropathogens by agar
well diffusion method.
2.12.4. Assimilation of carbon sources
The utilization of various carbon sources of different endophytic
actinomycetes strains were performed by the method was followed
by Ref. [38] and recommended in International Streptomyces Proj-
ect Medium (ISP2). The stock solution containing the 10con-
centration of different carbon sources i.e., xylose, inositol, sucrose,
raffinose, fructose, rhamnose and mannitol were prepared on
double distilled water and filtered by using 0.22
m
m pore size
membrane filter and stored at 4
C for further use. The strains were
streaked with 1% of carbon sources (1%) containing sterile ISP2
medium and the plates were incubated at 28
C for 7e15 days. The
growth of the actinomycetes were identified depending on the
utilization of carbon sources and positive growth of the results
were called as positive (P), if no growth occurs it is referred as
negative (N). The influence of active carbon compounds growth of
the active strains was used to check their antimicrobial activity by
well diffusion method.
2.12.5. Assimilation of nitrogen sources
The utilization of various nitrogen sources of different endo-
phytic actinomycetes strains were studied by the method was fol-
lowed by Ref. [39] and recommended in International Streptomyces
Project medium (ISP2). The stock solution containing the 10
concentration of various nitrogen sources i.e., L-Argine, L-Coralline,
L-Histidine, L-Glysine, L-Lysine and L-Proline were prepared with
double distilled water and filtered by using 0.22
m
m pore size
membrane filter at 4
C for future use. Strains were streaked with
1% of the nitrogen sources containing sterile ISP2 medium at 28
C
for 7e15 days. The growth of the actinomycetes were identified
depending on the uptake of nitrogen sources and the results were
called as positive reactions (P), if no growth occurred they were
referred as negative reaction(N). The influences of active nitrogen
compounds growth of strains were used to check antimicrobial
activity by well diffusion method.
3. Statistical analysis
The experiments were carried out independently in triplicate
with pooled samples of biological replicates. Statistical analysis was
performed using SPSS. Values were expressed as mean þSD. A
Duncan-ANOVA test with a p-value of 0.001 being highlysignificant
and to compare the parameters between the groups [40].
4. Results
4.1. Isolation and identification of endophytic actinomycetes
The healthy leaves of two brown algae (Fig. 1) were collected
from Gulf of Mannar region Rameswaram, Tamil Nadu, South East
coast of India. In validation, no microbial colonies were observed in
the ISP 2 plates and the result noticed that the sterilization was
good. After validation, the 100 pure ribbons like powdery white
color colonies of endophytic actinomycete were isolated from the
two algal samples grow on SCA medium and AIA medium respec-
tively. The isolated strains were recorded in (Fig. 2). Approximately
40% (40 isolates) of the endophytic actinomycetes strains were
observed with good antimicrobial activity against various clinical
pathogens. These active strains were further studied for the pro-
duction of bioactive compounds and the strains were identified by
gram staining, biochemical, physiological characterization, and
genomic studies. From validation, the result proved the isolated
actinomycetes were recovered from internal tissues of the algae
(Data not Shown).
4.2. Primary screening and antagonistic activity of isolated
endophytic actinomycetes against MDRS of UTI infection
The multi drug resistant effect of pathogens was screened
against fourth generation of cephalosporin (Ceftazidime) and the
result confirms the pathogens were multidrug resistant using disc
diffusion agar well diffusion method (SF. 1). In the primary
screening, the antagonistic activities of 40 isolated strains were
determined for antimicrobial activity against various multi drug
resistant uropathogens. Among the 40 strains, 10 strains (first five
strains from Turbinaria ornata, and second five strains from
(Sargassum wightii)) were showed comparatively better antago-
nistic activity (Table 1). They also showed minor discrepancy in
relation to different strains and test organisms. Interestingly,
Nocardiopsis sp. GRG1 (KT235640) showed relatively better anti-
bacterial activity against all isolated UTI pathogens (20) than other
nine strains and this Nocardiopsis sp. GRG1 (KT235640) strain was
chosen for further studies.
4.3. Extraction and antimicrobial activity of secondary metabolites
from Nocardiopsis sp. GRG1 (KT235640)
The potential strain of Nocardiopsis sp. GRG1 (KT235640)
showed excellent antibacterial activity was selected and inoculated
into starch casein broth for 4e7daysat28
C. After 7days, the
secondary metabolites were extracted by different polarity solvents
and the extracts were further screened for antimicrobial activity
against multi drug resistant strains of UTI pathogens. However, only
ethyl acetate extract (SF. 2) of the Nocardiopsis sp. GRG1
(KT235640) has shown good activity against all the test pathogens
except CoN. Staphylococci. The zone of inhibition was18 mm for
E. coli, 15 mm for P. aeroginosa, 14 mm for K. pneumonia, 13 mm for
Enterobacter, 15 mm for S. aereus and 30 mm for P. mirabilis were
observed (Fig. 3A). No zone of inhibition was observed in the
control well. When compared with other solvents, ethyl acetate
extract of Nocardiopsis sp. GRG1 (KT235640) showed good activity
and the zone of inhibition were presented in (Table 2).
4.4. Minimum inhibitory concentration (MIC)
Minimum Inhibitory Concentration (MIC) is referred as the
highest dilution or least concentration of the extract that inhibit
growth of organisms. MIC is an important parameter that helps to
determine the activity of newly discovered compounds against
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e114
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
various types of pathogens. The ethyl acetate extract of the
Nocardiopsis sp. GRG1 (KT235640) showed 80% inhibition against
MDR strains of P.mirabilis, E.coli, P.aeruginosa, S.aereus, Enterococcus
at and 73% inhibition against K.pneumonia and 77% inhibition
against CoN. Staphylococci were observed at a concentration of
100
m
g/ml. The treatment of Nocardiopsis sp. GRG1 (KT235640) was
decreased the pathogenic effect in MDRS by concentration-
dependent. This extract revealed a maximum inhibition (78 and
80%) against most of the uropathogens (Fig. 3B) at the same con-
centration (100
m
g/ml). Hence, 100
m
g/ml was chosen for further
study. The statistical analysis [ST3] reveals that the extract was very
efficient against uropathogens by comparing between test patho-
gens and control.
4.5. Biochemical characterization of endophytic actinomycetes
isolates
The highly active principle of the Nocardiopsis sp. GRG1
(KT235640) was characterized by various biochemical tests with
broth and slant cultures. After 24 h incubation, the MR, citrate,
urease, catalase, oxidase were observed as positive as well as
Indole, VP, and H
2
S were observed as negative. [ST4].
4.6. Genomic studies of potential strains
The 16S rDNA sequence of the Nocardiopsis sp. GRG1 was pro-
cessed (GC content 57%) and deposited in the Genbank (NCBI) with
the Accession number: KT235640. The phylogenetic tree analysis
showed that the 347bp sequence has the highest homology (98.5%
identity) with the Nocardiopsis sp. GRG1 (KT235640) (Fig. 4).
4.7. Phenotypic characterization
The phenotypic characterizations of 10 active strains of endo-
phytic actinomycetes strains were studied with aerial mass color,
reverse side pigments, melanoid pigments, carbon, nitrogen
Fig. 1. Collection of brown Algae from Gulf of Mannar Region.
Fig. 2. Isolation of endophytic actinomycetes.
Table 1
Identification of endophytic actinomycetes.
S.No Strains Name of the organisms and accession number Antagonistic activity
1 GRG1 Nocardiopsis sp. GRG1 (KT235640) Good Activity
2 GRG 2 Nocardiopsis sp. GRG 2 (KT235641) Good Activity
3 GRG3 Nocardiopsis sp. GRG 3 (KT235642) No Activity
4 GRG 4 Submitted No Activity
5 GRG 5 Submitted Poor Activity
6 GRG 6 Submitted No activity
7 GRG 7 Submitted Good Activity
8 GRG 8 Submitted Poor Activity
9 GRG 9 Submitted No Activity
10 GRG 10 Submitted No Activity
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e11 5
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
sources and spore chain morphology. The identification of colors
(Fig. 5B- a.f) was recorded in (Table 3).
4.7.1. Aerial mass color
After 5e7 days of incubation of 10 active strains, the white and
grey color colonies were observed in all strains of the SCA plates
and the heavy spores of the mycelia growth was also observed in all
the SCA plates. These results demonstrate the common character-
istics of actinomycetes (Fig. 5A a.e).
4.7.2. Reverse side pigments
The isolated endophytic actinomycetes strain ofGRG2, GEG3,
GRG6 and GRG9 (Fig. 5B c.e) were produced yellow color pigmen-
tation and the growth was also called as a positive or distinctive
character. The other plates did not produce any pigmentation in
their growth and were called as a non-distinctive or negative
character.
4.7.3. Melanoid pigments
The strains of GRG1 and GRG2 were observed with greenish
Fig. 3. (A). Antimicrobial activity of Nocardiopsis sp. GRG1 (KT235640) against MDRS of uropathogens. (B). Percentage of inhibiton by Minimum Inhibition Concentration (MIC)
against Multi drug resistant strains (MDRs) of Uropathogens.
Table 2
Antimicrobial activity of Nocardiopsis sp. GRG1 (KT235640) against UTIs.
S.No Pathogens Ethyl acetate extract Methanol extract Chloroform extract Control (Ethyl acetate)
Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition (mm)
1 E.coli 18 6 10 e
2Proteus mirabilis, 24 14 9 e
3Pseudomonas aeruginosa 15 8 8 e
4Klebsiella pneumoniae 14 5 5 e
5Staphylocoous aureus 15 4 7 e
6Enterobacter 13 9 7 e
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Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
brown to black diffusible pigment production and the strains were
also called as positive producer. Rest of the other strains did not
produce any pigment (Fig. 5B a.b) in their nature and they were
called as negative producer. The positive and negative growth of
the substrate mycelium and aerial mycelium werealso studied with
all the strains and the results were recorded in above.
4.8. Optimization of endophytic actinomyctes
The effect of salinity, temperature and pH of the isolated strains
were evaluated by using various concentrations (0
m
g/ml, 5
m
g/ml,
10
m
g/ml, 15
m
g/ml, 20
m
g/ml, 25
m
g/ml, 30
m
g/ml, 35
m
g/ml, 40
m
g/
ml, 45
m
g/ml, 50
m
g/ml, 55
m
g/ml, 60
m
g/ml, 65
m
g/ml, 70
m
g/ml,
75
m
g/ml) respectively.
4.8.1. Effect of salinity
After 6e15 days of incubation, the isolated strains of GRG1,
GRG2, GRG4, GRG6, and GRG9 showed increase in growth and
turbidity at 5% concentration. Some strains were also grown in the
same concentration and moderate level at 7.5% concentration. None
of the strains were exhibited growth in minimum concentration of
5e7%. Among these isolates, the active strain of GRG1 showed
better antimicrobial activity against uropathogen than other iso-
lates at the concentration of 5% [ST5].The other concentrations of
this strain did not produce better activity (Fig. 6A).
4.8.2. Effect of temperature
The effect of temperature is one of the most important factors
used for the identification of actinomycetes. A different type of
endophytic actinomycetes was isolated depends on the tempera-
ture level. Among the10 isolates, all the strains were easily grown in
the temperature of 25
o
C-45
C(SF. 3A) and some strains were able
to grow at 55
C. The strains were observed with moderate and fair
level of the growth at 40
o
C-50
C. Few strains (GRG8, GRG9) were
did not grow in the temperature of 55
C [ST6]. The excellent ac-
tivity of Nocardiopsis sp. GRG1 (KT235640) extract was observed at
30
C and other temperatures were not perfect for production of
potential antimicrobial compounds the picture was shown in
(Fig. 6B).
4.8.3. Effect of pH
All the isolated strains were identified using various pH levels
(4e9). Almost all the strains were easily grown at the level of pH
(5e8) (SF. 3B). Few strains were not grown at pH 4. Hence, the
potential antimicrobial compounds of Nocardiopsis sp. GRG1
(KT235640) against uropathogens were synthesized at pH7 [ST7]
and the acetic pH has not able to produce any active metabolites.
The picture was shown in (Fig. 6C).
4.8.4. Assimilation of carbon sources by the endophytic
actinomycetes isolates
The utilization of carbon compounds indicates a good source of
energy for all strains of endophytic actinomycetes. After 5 days of
incubation, the fructose was determined as a major carbon com-
pound for all the strains of endophytic actinomycetes. Because, all
the strains were able to grown in fructose and absence in mannitol
were observed (SF. 4A). The results were compared with positive as
well as negative result of carbon utilization ability, the fructose
showed highly positive to all isolates and the mannitol showed
negative to all isolates [ST8]. Hence, the excellent activity of
Nocardiopsis sp. GRG1 (KT235640) extract was observed in fructose
containing broth. The image was shown in (Fig. 6D).
4.8.5. Assimilation of nitrogen sources by the endophytic
actinomycetes isolates
The ability to utilize to various nitrogen compounds is a source
of energy for the isolated strains of endophytic actinomycetes and
the isolates were performed by ISP-2. After 5 days of incubation, the
L-Aspergine was determined as a major nitrogen compound for all
the strains of endophytic actinomycetes (SF. 4B). Because, almost all
isolated strains were grown well in L-Aspergine, and L-Proline was
not suitable for all the strains and the compound was identified as
low level. The growth were compared with positive and negative
control, the L-Arginine was observed as highly positive to all the
isolates and L-Proline was observed as negative for all the isolates
and the results were recorded in [ST9]. Hence, the excellent activity
of Nocardiopsis sp. GRG1 (KT235640) extract was determined in L-
Aspargine containing broth. The picture was shown in (Fig. 6E).
Furthermore, the standard deviation of MIC, Temperature, pH,
Carbon assimilation, Nitrogen assimilation were clearly indicates
the statistically significant (p <0.001) of endophytic actinomycetes
and the value of correlation of MIC was noticed in table [ST 10].
5. Discussion
In our study, a total of 100 strains of endophytic actinomycetes
were isolated from two brown algae. Among the 100 isolates, 40
isolates were more effective against clinical pathogens and 10
active strains were sequenced based on the respective order of
broad spectrum of antimicrobial activity against uropathogens.
The antagonistic activities of GRG1, GRG2, GRG3, GRG4, GRG5,
GRG6, GRG7, GRG8, GRG9 and GRG10 were identified in SC agar
plates. Among these 10 strains, Nocardiopsis sp. GRG1 (KT235640)
showed very good activity against gram negative bacteria than
gram positive bacteria of UTI pathogens. The ethyl acetate extract of
Nocardiopsis sp. GRG1 (KT235640) showed excellent activity and
high zone of inhibition against P. mirabilis (30 mm), P.aeroginosa
(15 mm), E.coli (18 mm), S. aereus (15 mm), K. pneumonia (14 mm)
and the minimum zone of inhibition against Enterobacter sp
(13 mm) were determined. The methanol extract showed the ac-
tivity against P. mirabilis (14 mm), P. aeroginosa (8 mm), E.coli
(6 mm), S.s aereus (4 mm), K. pneumonia (5 mm) and Enterobacter
sp (6 mm) and Chloroform extract showed the activity against
P. mirabilis (9 mm), P. aeroginosa (8 mm), E.coli (10 mm), S. aereus
(7 mm), K. pneumonia (5 mm) and Enterobacter sp (7 mm) were
observed [39]. Hence, the ethyl acetate extract was observed with
better activity compared with other solvent extract. Our results
were in accordance with the earlier findings of [20,41] and reported
that Nocardia brasiliensis PTCC 1422 has showed significant anti-
microbial activity against P.mirabilis (9 mm), P. aeroginosa (12 mm),
E.coli (17 mm), K. pnemoniae (15 mm).
At the3rd day of incubation, the maximum and minimum
antimicrobial activity of Nocardiopsis sp. GRG1 (KT235640) showed
better antimicrobial activity against Proteus mirabilis (16 mm),
Fig. 4. Phylogenetic analysis of Nocardiopsis sp GRG1 (KT235640).
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e11 7
Please cite this article in press as: G. Rajivgandhi, et al., Molecular characterization and antibacterial effect of endophytic actinomycetes
Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
Fig. 5. (a). Aerial mass culture of endophytic actinomycetes strains (a.e). (b). Phenotypic characterizations of isolated endophytic actinomycetes strains (aee).
Table 3
Phenotypic characterization of endophytic actinomycetes isolates.
S.No Isolates Aerial Mass color Melanoid pigments Reverse side pigments Aerial mycelium Substrate mycelium
1Nocardiopsis sp. GRG1 (KT235640) W N p P P
2Nocardiopsis sp. GRG 2 (KT235641) W P P P P
3Nocardiopsis sp. GRG 3 (KT235642) G P P P P
4 GRG4 G N N P P
5 GRG5 W N N P P
6 GRG6 W P N P P
7 GRG7 W P P P P
8 GRG8 G N N P P
9 GRG9 G P N P P
10 GRG10 W N P P P
P: Positive Growth, N: Negative Growth, W: White, G: Grey.
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Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
E. coli (15 mm) and P. aeroginosa (14 mm). At the 7th day of incu-
bation, the growth was increased and reached with excellent ac-
tivity against these pathogens were observed. Nocardiopsis sp.
GRG1 (KT235640) and Nocardia brasiliensis PTCC 1422 showed
similar activity was observed at the 7th day of incubation. Our re-
sults confirm the study of [42].
MIC was used to determine the inhibition ranges of pathogen at
various concentrations. MIC of a Nocardiopsis sp. GRG1 (KT235640)
extracts has reduced the inhibition up to 77%e80% against
P. mirabilis, E.coli, S.aureus, Enterococcus and K.Pneumonia, CoN
Staphylococcus respectively. Our result confirms that the antimi-
crobial activity at the concentration of 100
m
g/ml has showed better
activity against UTI pathogens. Hence, 100
m
g/ml was used for
further analysis [43].
The aerial mass of the strains were observed with white color
powdery growth (GRG1, GRG2, GRG5, GRG6, GRG7 and GRG10) in
almost all the strains and few strains were showed whitest grey
color (GRG3, GRG4, GRG8, and GRG9). These results were correlated
with their finding [44,45].
Further, we have studied the effects of salinity by using various
concentrations of NaCl with isolated strains. Almost all the strains
were observed as negative at the concentration of 5e25%. Very few
strains were shown as positive at the concentration of 5e7.5%
because of their salt nature (low salt condition). Our results were
similar to their work of [45,46].
The production of potential antimicrobial metabolites were
depends on the temperature. Here, the determination of Nocar-
diopsis sp. GRG1 (KT235640) has showed excellent activity at 30
C
and no activity was observed at 5
o
C-10
C. The isolated strain
showed excellent activity against P.mirabilis (14 mm), E.coli
(16 mm), P. aeroginosa (14 mm), S. aureus (13 mm) at 30
C. Our
results confirm the study of (48), that Streptomyces afghaniensis
VPTS3-1 was observed as highly active against P.Valgaris (20 mm)
and B.subtilis (12 mm) at 30
C.
Studying various levels of pH is one of the important parameter
was used to synthesize the new secondary metabolites and anti-
biotic production [47].
In our study, at optimum pH-7, the potential secondary me-
tabolites has produced excellent zone of inhibition against P.mir-
abilis (16 mm), E.coli (14 mm), P. aeroginosa (15 mm), S.aureus (14)
Fig. 6. (a). Sodium Chloride Tolerance on various endophytic actinomycetes Growth. (b). Effect of Different Temperature on various endophytic actinomycetes growth. (c). Effect of
Different pH level on various endophytic actinomycetes growth. (d). Carbon utilization of isolated endophytic actinomycetes. (e). Nitrogen utilization of isolated endophytic
actinomycetes.
G. Rajivgandhi et al. / Bioactive Materials xxx (2016) 1e11 9
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Nocardiopsis sp. GRG1 (KT235640) from brown algae against MDR strains of uropathogens, Bioactive Materials (2016), http://dx.doi.org/
10.1016/j.bioactmat.2016.11.002
and Enterobacter (15 mm). Because, acidic condition is not suitable
for production of potential antimicrobial compounds. The Nocar-
diopsis spp. TE1 and APA1 were grown well and produced potential
antimicrobial compounds in optimum pH7. Our results were
correlated with their findings of [19,48].
The carbon and nitrogen compounds utilization was one of the
most important factors for identification of endophytic actinomy-
cetes. Out of 10 active isolates, we have determined only fructose
and raffinose are the main sources utilized by the strains GRG1,
GRG2, GRG3, GRG5, GRG6, GRG7 and GRG10 as highly positive.
GRG4, GRG8, GRG9 were observed as negative and Mannitol was
found negative in most of the strains. For nitrogen utilization, the
isolated strains were easily grown with L-Aspargin and most of the
strains were not grown with L-proline. The similar results were
observed with the nitrogen utilization ability were recorded [49].
But, the results were in comparison with [44], which was totally
different and their work on the mannitol was the most assimilated
carbon sources by all strains of the actinomycetes and the arabinose
was least carbon sources utilized by all strains of actinomycetes
[38].
6. Conclusion
Our findings, Nocardiopsis sp. GRG1 (KT235640) act as a major
source of novel antibiotics against various types of pathogens
mainly UTIs. Therefore, isolation, characterization and study of
Nocardiopsis sp. GRG1 (KT235640) has been useful in discovery of
novel compounds. The nature of marine environment, low salinity,
optimum pH, high temperature and carbon-nitrogen content in-
fluences Nocardiopsis sp. GRG1 (KT235640) revealed tremendous
antimicrobial activity against Proteus mirabilis and Pseudomonas
aeroginosa and minimum zone of inhibition against E.coli, Staphy-
lococcus aereus, Kiebsiella pneumonia. This study helps in designing
drugs against multi drug resistant strains of urinary tract infections.
Acknowledgment
The authors would like to thank for Mr. T.Santhanakrishnan,
Geological and Biostatistical Laboratory, Department of Marine
Science, Bharathidasan University for statistical analysis discussion.
We gratitude to the Bharathidasan University, Tiruchirappalli-24
for University Research Fellowship (URF) (Ref. No. 05441/URF/K7/
2013).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.bioactmat.2016.11.002.
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10.1016/j.bioactmat.2016.11.002
